The inter-conversion of telomeres between different functional states, defined by the composition of the bound proteins, is the fundamental step that dictates whether the chromosome end is recognized as a bona-fide end or incorrectly as a double-strand break. In recent years it has become evident that motor proteins (e.g. helicases) play a role in telomere regulation. Even in yeast, one of the best-studied model systems, how helicases function in telomere regulation is not fully understood. For example, it is not clear whether the major function of these motor proteins originate from their helicase activity and thus their ability to unwind double-stranded nucleic acid or from their ability to translocate along DNA and remove bound proteins. In S.cerevisiae, Pif1 helicase affects telomere function via displacement of the telomerase, the specialized reverse transcriptase responsible for extension of the G-rich strand of the telomere. A second helicase, Rrm3, has been implicated in telomere function. Rrm3 facilitates replication fork progression at >1000 chromosomal sites, including telomeres. A possible function of Rrm3 in replication fork progression at telomeres is the displacement of Rap1 bound to the duplex region of the telomere. Knowledge of the intrinsic biochemical properties of Pif1 and Rrm3 and how they are employed/altered to carry out their functions is necessary to elucidate how these motor proteins participate in telomere regulation. In Specific Aim 1 we will: A) Study the allosteric modulation by nucleotide cofactors of Pif1-DNA interactions. B) Determine which oligomeric state of Pif1 is responsible for its activity as a helicase or a translocase and how Pif1 catalyzes these activities. C) Test whether Pif1 inhibition of the telomerase originates from its helicase activity or its ability to displace subunits of the telomerase complex. In Specific Aim 2 we will: A) Test our hypothesis that the ATPase and helicase activities of Rrm3 are regulated by a sequence/domain within its N-terminal region. B) Test the hypothesis that PCNA binding to Rrm3 relieves the regulatory function of its N-terminal region. C) Test the hypothesis that interaction with PCNA leads to an active Rrm3 that is able to displace Rap1 from the telomere. These studies will allow us to determine how Pif1 and Rrm3 function and will provide unique insight on how these enzymes participate in telomere/telomerase regulation. We expect to define whether oligomerization of Pif1 leads to separation of its functions and thus to its specific role at telomeres. Also, we expect to determine how the activities of Rrm3 can be regulated to allow removal of proteins bound to double-stranded region of the telomere, thus facilitating replication of these sites.